The overall goal of this procedure is to observe larval stage Drosophila crawling in a linear agarose channel to study the development of neural circuits. This method can help answer key questions in the fields of neuro ethology and developmental biology, such as understanding the neural codes that generate motor patterns and identifying key genetic and cellular processes that scope neuronal circuits for movement control. The main advantage of this technique is that it restricts the larval behavior repertoire so that the researchers can study a single multi pattern in detail.
Demonstrating the procedure will be Zarion Marshall a technician from my laboratory. First it is important to assess the larva's health. Three to four days before recording, replace the collection plate of the adult cage.
Proceed to do this daily and at the same time of day. Count the eggs and newly hatched larvae on the plate. The expected number of eggs is between 500 and 2, 000.
Adjust the number of adults in the cage as needed. We examine the plate 24 hours later to determine whether the larvae are healthy. Most of the eggs should have hatched into larvae, and the larvae should have crawled into the yeast paste.
Signs of poor health include a large number of unhatched eggs, dead larval bodies away from the yeast paste, and larvae with unusually dark patches in the abdomen which is an indicator of an immune reaction. If the larvae appear unhealthy, change the cage using freshly prepared plates, and check the adults genotype. For the experiment collect newly hatched second instar larvae from one to two day old plates.
All these stages can be used in the analysis. For this protocol, have a linear channel PDMS casting mold. Clean the casting mold with isopropyl alcohol.
Once it is dried, place the mold into a ten centimeter lid or similar container. Then fill the mold with three percent agarose cooled to 55 degrees Celsius. Pour the agarose carefully to avoid trapping air bubbles in the mold.
The mold should be just covered by the agarose. Once solidified, remove the agarose channels from the mold. Trim the edges of the disc shaped channels, and then immerse the channels in water.
They can be stored in water at four degrees Celsius for up to seven days. Before using the channels, allow them to warm to room temperature. Select a channel that matches the width of the larva.
The channels range from 100 to 300 microns in width and are all 150 microns deep. Then use a clean razor blade to cut a single channel from the prepared disc. With forceps transfer the channel to an appropriately sized glass cover slip or slide with the grooved side up.
Next, use fine forceps to gently pick up a larva without squeezing it. A finely tipped paintbrush also works well. If squeezed, pain reflexes such as rolling or hunching may dominate the larva's behavior in the channel.
Now wash the larva by briefly submerging it in water, and then place it into the cut channel gently teasing it into position. Once in the channel, adjust the orientation of the larva to make the structure of interest easy to image. Then place one or two drops of water at either end of the channel to fill the channel with water.
If any air bubbles get trapped, gently lift the sides of the channel to remove them. To further adjust the orientation of the larva, gently nudge the channel to roll the larva around. The larva is now ready to be imaged.
Larvae in linear channels perform sustained bouts of rhythmic crawling. Fluorescent probes are easily examined. For example, larvae expressing GFP in their neurons from the GAL4 driver show dynamic changes in fluorescent intensities emanating from the nerve cord during the crawl cycle.
The CNS moves forward at nearly the same time as the larval head and tail as a wave of muscle contraction passes along the body axis, the CNS moves in and out of the plane of focus, causing the fluorescence of the nerve cord to change. The changes in fluorescence were quantified from three crawl strides for three larvae. The dynamics of the nerve cord fluorescence over the stride cycle followed a reproduce able pattern that could be used for phenotyping.
After watching this video, you should have an understanding of how to collect larvae for behavior, to prepare agarose channels, and to load the larvae into the channels to visualize the linear crawling behavior. While attempting this procedure, it is important to remember to handle the larvae gently.
